We are investigating the mechanisms whereby steroid and polypeptide hormones stimulate growth and specific protein synthesis in human breast cancer both in tissue culture model systems we have established and in clinical settings. A. We are studying intracellular pharmacokinetics of estrogen and anti-estrogen metabolism and efflux from human breast cancer cells using perfusion systems. These studies have led to new insights into hormone receptor interactions with the genome. Specifically, we have discovered that intranuclear estrogen receptors are changed over time to a less easily extractable form associated with the onset of steroid induced effects. This "processed" receptor appears tightly bound to DNA, is extractable by nuclease digestion and may be the proximate receptor form involved in gene regulation. B. We are studying the detailed regulation of DNA synthesis in human breast cancer cells and as such have developed ways for accurately quantifying total DNA synthesis together with the scavenger and denovo pathways of pyrimidine biosynthesis. C. We have developed a soft agar cloning technique which has permitted the development of clones of antiestrogen resistant variant (putative mutant) cell lines derived from hormone dependent wild typed cells. These variant cells are currently being analyzed biochemically and via somatic cell hybridization.